Name: DNA-FISH Metaphase Spreads to Distinguish Extrachromosomal DNA from Homogeneously Staining Regions in Human Cancer Cell Lines (Video) | JoVE Journal
Uploaded: 2026-08-04T13:35:00.000+00:00
Duration: 8 min 21 s
Description: Learn DNA-FISH metaphase spreads to distinguish extrachromosomal DNA from homogeneously staining regions in human cancer cell lines. This protocol enables single-cell resolution analysis of oncogene amplification structures.

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5 minutes

This work was supported by NIH R00HG011467 and CPRIT RR23004 to G.S.E. Imaging for this project was supported by the Integrated Microscopy Core at Baylor College of Medicine with funding from the NIH (P30 DK056338, P30 CA125123, S10OD030414). The authors thank Dr. Pawel Stankiewicz for his advice early on. The authors also thank Dr. Paul Mischel (Stanford University) for providing the GBM39ec and GBM39hsr cell lines.

All human cell culture procedures were performed in compliance with institutional guidelines.
1. Arrest cells in metaphase using Colcemid (N-methyl-N-deacetyl-colchicine)
<ol>
	<li>Begin with approximately 2.0 &#215; 106&#8211;2.0 &#215; 107 cancer cells or other cultured cells. Too few cells will yield fewer metaphase spreads, whereas too many cells may result in insufficient fixation.</li>
	<li>Determine the appropriate Colcemid incubation time for the experimental system. Incubation time affects the number of metaphase spreads obtained and chromosome length, and optimal conditions vary among cell types. 
	NOTE: Shorter incubation times result in fewer cells arrested in metaphase at the time of collection. Longer incubation times produce more condensed chromosomes with shorter arms, making analysis more difficult. For cancer cell lines with an approximate doubling time of 24 h, incubation periods ranging from 45 min to overnight produce suitable metaphase spreads. Optimize the incubation time for cells with slower doubling times. A 2 h incubation is generally sufficient to produce an adequate number of metaphase spreads with chromosome lengths suitable for analysis.</li>
	<li>Calculate the amount of Colcemid required to achieve a final concentration of 10 &#181;L/mL in the appropriate cell culture medium. 
	NOTE: Maintain cells in logarithmic growth by passaging or seeding them at least 24 h before metaphase arrest. Treat cells with Colcemid when cultures reach 70%&#8211;90% confluence.</li>
	<li>Dilute the Colcemid stock solution 1:1 with sterile ultrapure water. Add the appropriate volume to the culture and incubate for 45 min to overnight in a cell culture incubator.</li>
</ol>
2. Harvest cells
<ol>
	<li>Prepare fresh Carnoy&#39;s fixative (3:1 methanol: acetic acid). Keep the fixative on ice throughout the fixation procedure.</li>
	<li>Prepare a fresh 0.075 M KCl solution and warm it in a 37 &#176;C water bath.</li>
	<li>Collect the cells as a pellet and record the cell count. Use this value to calculate the final resuspension volume. 
	NOTE: Detach adherent cells by trypsinization. Transfer suspension cells directly into 15 mL centrifuge tubes. Dispose of Colcemid and Colcemid-containing media according to institutional hazardous waste guidelines.</li>
	<li>Wash the cells with PBS.</li>
	<li>Gently resuspend the cells in 10 mL of warm 0.075 M KCl and mix by gentle inversion. This hypotonic treatment swells the cells, allowing them to rupture and disperse chromosomes during slide preparation.</li>
	<li>Incubate the cells in a 37 &#176;C water bath for 30 min.</li>
	<li>Slowly add 2 mL of ice-cold Carnoy&#39;s fixative while gently mixing the suspension by inversion.</li>
	<li>Allow the cells to stand for 5 min.</li>
	<li>Centrifuge at 500 &#215; g for 5 min at room temperature (RT).</li>
	<li>Carefully aspirate the supernatant or remove it with a pipette.</li>
	<li>Slowly add 10 mL of ice-cold Carnoy&#39;s fixative in 0.5 mL increments while gently mixing after each addition.</li>
	<li>Allow the cells to stand for 10 min at RT.</li>
	<li>Centrifuge at 650 &#215; g for 5 min at RT.</li>
	<li>Repeat steps 2.10&#8211;2.13 twice</li>
	<li>During the final wash, leave the cells suspended in Carnoy&#39;s fixative. 
	PAUSE: Store the fixed cell suspension at &#8722;20 &#176;C or proceed immediately to Section 3. 
	NOTE: For samples containing fewer than approximately 3 &#215; 106 cells, perform fixation in 1.5 mL microcentrifuge tubes instead of larger conical tubes. Reduce the volumes of 0.075 M KCl and Carnoy&#39;s fixative 10-fold while maintaining all incubation times, temperatures, and centrifugation conditions.</li>
</ol>
3. Tabletop dropping
<ol>
	<li>Retrieve the sample from &#8722;20 &#176;C and thaw it to RT.</li>
	<li>Centrifuge at 800 &#215; g for 7 min at RT.</li>
	<li>Label the glass slides and place them on ice.</li>
	<li>Using the cell count obtained before fixation, calculate the volume of Carnoy&#39;s fixative required to resuspend the pellet at a final concentration of approximately 5 &#215; 106 cells/mL. After centrifugation, carefully remove the supernatant with a pipette, leaving the calculated volume of fixative above the pellet.</li>
	<li>Resuspend the fixed cells in Carnoy&#39;s fixative using a P1000 pipette.</li>
	<li>Transfer approximately 300 &#181;L of the fixed-cell suspension into a 1.5 mL microcentrifuge tube.</li>
	<li>Thoroughly resuspend the sample using a P200 pipette by pipetting up and down several times.</li>
	<li>Aspirate 60 &#181;L of the resuspended cells using a P200 pipette.</li>
	<li>Hold the pipette tip approximately 1 m above the glass slide on ice and aim for the center of the slide.</li>
	<li>Slowly dispense one drop of the cell suspension onto the slide. 
	NOTE: Mark the drop location on the edge of the slide with an ethanol-resistant marker to facilitate identification during subsequent steps.</li>
	<li>Allow the cells to dry on ice for 10 min.</li>
	<li>Place the slides on a slide incubator at 80 &#176;C for 10 min.</li>
	<li>Remove the slides with tongs and allow them to cool to the touch.</li>
	<li>Examine the slides using a phase-contrast microscope with a 10&#215; or 20&#215; objective. Confirm the presence of multiple well-dispersed metaphase spreads without excessive overlap.</li>
</ol>
4. Pretreat the slide
<ol>
	<li>Prepare Coplin jars for the wash steps
	<ol>
		<li>Prepare each reagent fresh: 70% acetic acid (40 mL), PBS (40 mL), PBS (45 mL), 70% ethanol (40 mL), 85% ethanol (40 mL), and 100% ethanol (40 mL).</li>
	</ol>
	</li>
	<li>Place the glass slides in 70% acetic acid for 2 min.</li>
	<li>Place the glass slides in 40 mL of PBS for 30 s.</li>
	<li>Place the glass slides in 45 mL of PBS for 5 min.</li>
	<li>Dehydrate the glass slides sequentially in 70%, 85%, and 100% ethanol for 1 min each.</li>
	<li>Air-dry the slides at RT for 10 min.</li>
</ol>
5. Prepare the probe
NOTE: This protocol uses commercially available locus-specific DNA-FISH probes that hybridize to a genomic region of interest.
<ol>
	<li>Select a probe targeting the amplified locus to be examined. Thaw the probe and hybridization buffer according to the manufacturer&#39;s instructions. Allow the probe to reach RT before use.</li>
	<li>Prepare a probe mixture containing 1 &#181;L of probe and 4 &#181;L of hybridization buffer for each glass slide. 
	NOTE: The optimal probe concentration may depend on the manufacturer and the probe. The concentration specified here has been optimized as per the probes used in this study. However, optimize the probe concentration for each new locus.</li>
	<li>Apply 5 &#181;L of the probe/hybridization buffer mixture to the center of each drop location on the glass slide. 
	NOTE: Probes are light-sensitive. Minimize light exposure throughout the remaining procedure by storing the slides in a dark drawer between steps and while processing other slides.</li>
	<li>Carefully place a coverslip over the sample.</li>
	<li>Seal the edges of the coverslip with rubber cement.</li>
	<li>Place the glass slides in a humid chamber.</li>
	<li>Hybridize the probe according to the manufacturer&#39;s protocol. Allow hybridization to proceed overnight (12&#8211;18 h).</li>
</ol>
6. Post-hybridization washes, counterstaining, mounting, and imaging
<ol>
	<li>Prepare Coplin jars for the wash steps
	<ol>
		<li>Prepare each reagent fresh: 2&#215; SSC (40 mL), 0.4&#215; SSC (40 mL), 70% ethanol (40 mL), 85% ethanol (40 mL), 100% ethanol (40 mL), and PBS (45 mL).</li>
		<li>Prepare and store SSC as a 20&#215; stock solution (3 M NaCl, 0.3 M sodium citrate, pH 7.0). To prepare 1 L, dissolve 175.3 g NaCl and 88.2 g sodium citrate in 1 L of ultrapure water. Dilute the stock to prepare 2&#215; SSC and 0.4&#215; SSC working solutions as needed.</li>
	</ol>
	</li>
	<li>Warm the 0.4&#215; SSC solution to 72 &#176;C in a water bath.</li>
	<li>Remove the glass slides from the humid chamber and carefully remove the rubber cement.</li>
	<li>Place the glass slides in 2&#215; SSC for 2 min at RT. Carefully remove the coverslips.</li>
	<li>Place the glass slides in prewarmed 0.4&#215; SSC at 72 &#176;C for 2 min.</li>
	<li>Place the glass slides in 2&#215; SSC for 1 min at RT.</li>
	<li>Dehydrate the glass slides sequentially in 70%, 85%, and 100% ethanol for 1 min each.</li>
	<li>Air-dry the slides at RT for 10 min in the dark.</li>
	<li>Dilute 1 mg/mL DAPI 1:1000 in PBS to a final volume of 1 mL. 
	NOTE: This aliquot can be stored at 4 &#176;C for up to 1 week.</li>
	<li>Pipette 10 &#181;L of the diluted DAPI onto each glass slide at the marked drop location.</li>
	<li>Carefully place a coverslip over the sample to ensure the DAPI solution spreads evenly.</li>
	<li>Incubate the slides in the dark for 5&#8211;10 min.</li>
	<li>Place the glass slides in PBS for 30 s.</li>
	<li>Remove the coverslips.</li>
	<li>Place the glass slides in PBS for 2 min.</li>
	<li>Dehydrate the glass slides by briefly dipping them in 70%, 85%, and 100% ethanol for approximately 10 s each.</li>
	<li>Air-dry the slides at RT for 10 min in the dark.</li>
	<li>Add 15 &#181;L of mounting medium to each glass slide, then place a coverslip on top.</li>
	<li>Allow the mounting medium to spread evenly, then gently blot away any excess.</li>
	<li>Seal the edges of the coverslip with nail polish.</li>
	<li>Allow the nail polish to dry for 15 min at RT in the dark. 
	PAUSE: Store the slides at 4 &#176;C for long-term storage.</li>
	<li>Image the slides. 
	NOTE: Images were acquired using an inverted fluorescence microscope with a 60&#215; oil-immersion objective.</li>
	<li>Collect multiple z-stacks for each fluorescence channel to capture signal throughout the full depth of the metaphase spread.</li>
	<li>Deconvolve the image stacks using a maximum-likelihood estimation algorithm to enhance signal clarity and reduce out-of-focus fluorescence.</li>
	<li>Process and analyze the final images in ImageJ, and select the optimal focal plane for each metaphase spread before downstream analysis.</li>
</ol>

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The authors declare no competing interests.

Despite their well-established advantages, DNA-FISH metaphase spreads are underused in the era of WGS. The physical organization of amplified DNA is functionally significant, as it can influence downstream biological behavior and therapeutic response61,62,63,64. Focally amplified oncogenes can exist on ecDNA or within HSRs, and these two amplification states are not functionally equivalent<sup ...

The human genome is organized not only by its sequence, but also by its structure, and changes in either sequence or structure can lead to cancer1,2,3,4. Oncogene amplification is present in approximately 40% of all tumors, where it contributes to tumor initiation, growth, metastasis, and drug resistance5. In some cases, amplified oncogenes are carried on extrachromosomal DNAs (ecDNAs, also called double minutes), which are circular, self-replicating, double-stranded DNA elements that reside outside of the linear chromosomes6,7,8,9. ecDNAs are hundreds of kilobases to megabases in size10,11. Due to their lack of centromeres, they segregate asymmetrically during mitosis, leading to as many as 100+ copies in a single nucleus12,13,14,15,16,17,18. This uneven segregation generates copy number variability across daughter cells, which drives rapid tumor evolution and poor patient outcome17,19.
ecDNAs exist in a dynamic relationship with homogeneously staining regions (HSRs) (Figure 1A)8,18. HSRs are focal, intrachromosomal amplifications that can arise through the reintegration of ecDNAs into a single locus in the genome8,10,18,20,21,22,23,24,25,26,27,28,29,30,31. These two states, ecDNAs and HSRs, can interconvert, revealing that oncogene amplification is structurally plastic24,25,32,33. This interconversion has therapeutic relevance because ecDNAs can transition to the HSR state in the presence of drugs32,33,34,35,36,37,38. Cells that undergo this transition to the HSR state then become resistant to the drug under therapeutic pressure33. HSRs also appear to be more frequent in malignant tumors than in benign tumors39. Despite their clinical relevance, no FDA-approved strategies target the HSR state, in part because methods to distinguish these amplification structures remain underutilized.
Although ecDNA and HSR states have distinct clinical consequences, current sequencing approaches cannot reliably distinguish them. Whole-genome sequencing (WGS) now identifies amplified regions with base-pair resolution40. WGS has been valuable in defining the sequence composition of ecDNAs and HSRs, but at the expense of spatial resolution. Unlike traditional cytogenetic approaches, WGS alone cannot determine whether an amplified sequence resides on ecDNAs or is integrated into a chromosome as an HSR41,42,43. As a result, methods that preserve the spatial context of amplification remain essential for understanding the biology of these distinct states.
For the past 40 years, DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads have served as the gold standard for identifying and distinguishing ecDNA and HSR amplification states42,44,45,46. In fact, ecDNAs and HSRs were first identified and characterized through metaphase-based cytogenetic analyses7,47. Before the widespread use of fluorescent probes, in situ hybridization was performed using radiolabeled DNA probes, which have also been used to visualize ecDNAs and HSRs48,49. As WGS gained in popularity, DNA-FISH metaphase spreads became less common. Many aspects of the workflow are scattered across the cytogenetic literature, and optimized protocols are typically retained within individual research groups or cytogenetics labs that treat the method as proprietary50,51. Here, we describe a DNA-FISH metaphase spread protocol to visually resolve amplification state at the single-cell level.

<table><tbody><tr><td>15-mL conical centrifuge</td><td>any source</td><td>NA</td><td>Centrifugation.&amp;nbsp;</td></tr><tr><td>15-mL conical tubes</td><td>any source</td><td>NA</td><td>Cell collection.</td></tr><tr><td>200 proof ethanol</td><td>any source</td><td>NA</td><td>Wash steps.&amp;nbsp;</td></tr><tr><td>Acetic Acid, glacial, 99.7%</td><td>Millipore Sigma</td><td>695092</td><td>Carnoys fixative and slide wash steps.</td></tr><tr><td>Antifade Mounting Medium</td><td>FisherScientific</td><td>NC9265087</td><td>Slide mounting after completed slide preparation</td></tr><tr><td>Biological safety cabinet with vaccum outlet</td><td>any source</td><td>NA</td><td>Chemical storage.</td></tr><tr><td>Colcemid (N-methyl-N-deacetyl-colchicine)</td><td>Millipore Sigma</td><td>10295892001</td><td>Binds tubulin, arrests cells in metaphase.</td></tr><tr><td>Coplin jars</td><td>any source</td><td>NA</td><td>Wash steps.&amp;nbsp;</td></tr><tr><td>DAPI (4&amp;prime;,6-diamidino-2-phenylindole)</td><td>Milipore Sigma</td><td>D8417-1MG</td><td>Fluorescent DNA stain.</td></tr><tr><td>EGFR/CON7 FISH probe&amp;nbsp;</td><td>Empire Genomics</td><td>EGFR-CHR07</td><td>Locus-specific fluorescent&amp;nbsp;probe.&amp;nbsp;</td></tr><tr><td>Elmer&#039;s rubber cement</td><td>any source</td><td>NA</td><td>Keep coverslide in place during overnight probe hybridization.</td></tr><tr><td>Ethanol resistant permanent marker</td><td>any source</td><td>NA</td><td>Slide labeling.</td></tr><tr><td>Fluorescent microscope</td><td>any source</td><td>NA</td><td>Slide imaging.&amp;nbsp;</td></tr><tr><td>Glass Cover Slip, 22 x 22mm</td><td>ThermoFisher</td><td>50-143-780</td><td>Probe hybridization on slide.</td></tr><tr><td>Glass Slide, precleaned</td><td>VWR</td><td>48311-703</td><td>To drop metaphase spreads and perform DNA-FISH.</td></tr><tr><td>Humidified CO2 Incubator</td><td>any source</td><td>NA</td><td>Live-cell incubation.</td></tr><tr><td>Hybridization Buffer</td><td>Empire Genomics</td><td>Hyb-Buffer</td><td>Buffer used with probe.&amp;nbsp;</td></tr><tr><td>Ice bucket</td><td>any source</td><td>NA</td><td>Keep Carnoy&#039;s fixative cold.&amp;nbsp;</td></tr><tr><td>KCl (2M)</td><td>ThermoFisher</td><td>AM9640G</td><td>Swell cells prior to fixation.&amp;nbsp;</td></tr><tr><td>Methanol, 99.9%, HPLC grade</td><td>Millipore Sigma</td><td>1060351000</td><td>For Carnoys fixative.&amp;nbsp;</td></tr><tr><td>Microcentrifuge</td><td>any source</td><td>NA</td><td>Centrifugation.&amp;nbsp;</td></tr><tr><td>Microcentrifuge tubes</td><td>any source</td><td>NA</td><td>For cells during fixation, and for probe preparation.&amp;nbsp;</td></tr><tr><td>MYC / SE 8 Probe</td><td>Leica</td><td>KI-10106</td><td>Locus-specific fluorescent&amp;nbsp;probe.&amp;nbsp;</td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010-023</td><td>Wash steps.&amp;nbsp;</td></tr><tr><td>Phase contrast microscope</td><td>any source</td><td>NA</td><td>Imaging of metaphase spreads immediately after dropping</td></tr><tr><td>Pipette tips</td><td>any source</td><td>NA</td><td>Pipetting.</td></tr><tr><td>Sodium chloride</td><td>Millipore Sigma</td><td>S9888</td><td>For SSC buffer.</td></tr><tr><td>Sodium citrate</td><td>Millipore Sigma</td><td>567446</td><td>For SSC buffer.</td></tr><tr><td>Sterile ultrapure water</td><td>any source</td><td>NA</td><td>Wash steps.&amp;nbsp;</td></tr><tr><td>ThermoBrite programmable slide incubator</td><td>any source</td><td>NA</td><td>Slide incubation.</td></tr><tr><td>Tongs</td><td>any source</td><td>NA</td><td>For slide wash steps.&amp;nbsp;</td></tr><tr><td>Water bath</td><td>any source</td><td>NA</td><td>For KCl incubation.&amp;nbsp;</td></tr></tbody></table>

dna-fish metaphase spreads to distinguish extrachromosomal dna from homogeneously staining regions in human cancer cell lines

DNA-FISH Metaphase Spreads to Distinguish Extrachromosomal DNA from Homogeneously Staining Regions in Human Cancer Cell Lines

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

This protocol (Figure 1B) yields clean, well-dispersed, intact metaphase chromosomes suitable for cytogenetic analysis, with chromosomes and DNA-FISH signals clearly visible by fluorescence microscopy. Using a brightfield/phase-contrast microscope at low magnification (10&#215; or 20&#215;) is an important quality-control step to verify that nuclei have burst and chromosomes have dispersed (Figure 2). Successful fixation and tabletop dropping should yield well-d...

We report a DNA-FISH metaphase spread protocol that visually detects locus copy number and location within the genome. This approach enables single-cell resolution of amplification states, specifically in cancer cell lines containing extrachromosomal DNA and homogeneously staining regions.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on ...

Our research focuses on determining whether amplified oncogenes reside on extrachromosomal DNA or within distinct chromosomally-integrated homogeneously staining regions. Sequencing fails to distinguish ecDNA from HSR amplification, so our protocol resolves this at single cell resolution. To begin, dilute the colcemid stock solution at a one-in-one ratio with sterile ultra-pure water.For the metaphase arrest, add 100 microliters of diluted colcemid solution to the COLO 320DM maintained in 10 milliliters of media at logarithmic growth at 70 to 90%confluence, and place them in a cell culture incubator for 45 minutes to overnight. Prepare a fresh Carnoy's fixative using a three-to-one ratio of methanol to acetic acid. Keep the Carnoy's fixative on ice throughout the fixation procedure.Prepare a fresh 0.075 molar potassium chloride solution and warm the solution in a 37 degrees Celsius water bath. After centrifugation and cell counting, wash the cells with PBS. Gently resuspend in 10 milliliters of the warm 0.075 molar potassium chloride solution and mix the suspension by gentle inversion.Incubate the cells in a 37 degrees Celsius water bath for 30 minutes. Slowly add two milliliters o

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Research

JoVE Journal

Biology

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis 

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Circulating tumor cells (CTC) isolation and characterization from the blood of cancer patients is a type of non-invasive liquid biopsy and associated with relapse and disease progression. This video describes the technique of DNA fluorescence-in-situ-hybridization (DNA FISH) on CTCs captured on a medical wire, to study a specific gene of interest.

3D DNA FISH: A Technique to Locate a Specific Gene on a Chromosome

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Robust 3D DNA FISH Using Directly Labeled Probes

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Learn DNA-FISH metaphase spreads to distinguish extrachromosomal DNA from homogeneously staining regions in human cancer cell lines. This protocol enables single-cell resolution analysis of oncogene amplification structures.

DNA-FISH Metaphase Spreads to Distinguish Extrachromosomal DNA from Homogeneously Staining Regions in Human Cancer Cell Lines

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